Expression and purification of recombinant methylated HBHA in Mycobacterium smegmatis

Giovanni Delogu, Alessandra Bua, Cinzia Pusceddu, Marcela Parra, Giovanni Fadda, Michael J. Brennan, Stefania Zanetti

Research output: Contribution to journalArticlepeer-review

Abstract

The Heparin-Binding Haemagglutinin (HBHA) is a mycobacterial adhesin involved in the dissemination of Mycobacterium tuberculosis from the site of primary infection and a potential candidate for the development of a new vaccine against tuberculosis. Methylation of HBHA is a novel post-translational event that imparts important immunological properties to the protein. Since recombinant HBHA expressed in Escherichia coli is not methylated, we investigated the possibility of producing recombinant methylated HBHA in fast growing mycobacteria for use in immunological and biochemical studies. The complete coding sequence of HBHA was cloned in the plasmid pMV206, under the control of a strong promoter (hsp60) or its own promoter. The constructs generated were electroporated into Mycobacterium smegmatis and the recombinant strains obtained were analyzed for the presence of the HBHA protein using the anti-HBHA monoclonal antibodies D2 and E4. Our results indicate that expression of high amounts of intact protein can be toxic for the mycobacteria, that methylated HBHA can be obtained in M. smegmatis only when using a promoter sequence weaker than hsp60 and that the expression of the complete structural gene is required in order to obtain methylated HBHA. We constructed a recombinant M. smegmatis strain (pMV3-38) that expresses a histidine-tagged methylated HBHA that can be easily purified. The use of fast-growing strains of M. smegmatis to obtain significant amounts of purified HBHA protein within a short timeframe, should be an effective strategy for the evaluation of a new HBHA-based vaccine candidate for tuberculosis.

Original languageEnglish
Pages (from-to)33-39
Number of pages7
JournalFEMS Microbiology Letters
Volume239
Issue number1
DOIs
Publication statusPublished - Oct 1 2004

Keywords

  • HBHA
  • Methylation
  • Mycobacteria
  • Mycobacterium
  • Protein expression
  • Tuberculosis
  • Vaccine

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Applied Microbiology and Biotechnology
  • Microbiology

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